Ratiometric Pulse–Chase Amidination Mass Spectrometry as a Probe of Biomolecular Complex Formation

• Published in: Analytical chemistry (Washington) Vol. 83; no. 23; pp. 9092 - 9099
• Main Authors: Chang, Feng-Ming James, Lauber, Matthew A, Running, William E, Reilly, James P, Giedroc, David P
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.12.2011
• Subjects: Amides - chemistry; Analytical chemistry; Bacillus subtilis - metabolism; Bacterial Proteins - metabolism; Binding sites; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid; Chymotrypsin - metabolism; Copper; Copper - chemistry; Exact sciences and technology; General, instrumentation; Kinetics; Lysine - chemistry; Mass spectrometry; Other chromatographic methods; Peptides - analysis; Proteins; Serine Endopeptidases - metabolism; Spectrometric and optical methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; High resolution; Peptides; Bacillus subtilis; Time; Matrix assisted laser desorption ionization; Bacillaceae; Probe; Electrospray; Bacillales; Bacteria; Copper I; Enzyme; Use; Liquid chromatography; Chemical sensor; Chemical ionization; Protein; Peptidases; Chemical modification; Lysine; Time of flight method; Residue; DNA; Mass spectrometry MS/MS; Transition; Hydrolases; Mass spectrometry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac202154r

Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse–chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (Δ = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.